Establishment Of Liver Cell Apoptosis Model And Evaluation Of Anti-apoptotic Activity Of Natural Compounds

The imbalance of hepatocyte apoptosis is an important factor leading toliver damage, especially acute liver failure or fulminant hepatitis. The regulation ofliver cell apoptosis to ameliorate liver inflammation and injury is considered as a newstrategy for the treatment of hepatitis. To investigate the pathogenesis of hepatitis andevaluate anti-hepatitis drug, it is necessary to establish the in vitro and in vivo modelsfor hepatitis and related liver disease. However, it is lack of a stable and reliablemodel of hepatocyte apoptosis so far, it causes a great difficulty for application of thenew hepatitis-treatment strategies and development of anti-hepatitis drugs. The aboveanalysis suggests that the establishment of hepatocyte apoptosis model has a greatsignificance for in-depth study of the pathogenic mechanism of hepatitis and thedevelopment of anti-hepatitis new drug.In this study, we established the D-Galactosamine (GalN)/lipopolysaccharide(LPS)-induced hepatocyte apoptosis model in mice and the GalN-induced cellapoptosis model in HL-7702hepatocytes, also explored the optimized conditions ofhepatocyte apoptosis and investigated the mechanisms involved in the process ofhepatocyte apoptosis. Furthermore, the effect of three natural dicaffeoylquinic acidson GalN-induced HL-7702hepatocyte apoptosis were also studied.Part1The establishment of liver apoptosis model in mice and its mechanismsinvolved in this processFor preparation of the mice with GalN/LPS-induced hepatocyte apoptosis, themice were injected intraperitoneally with the different concentrations of GalN(200-700mg/kg) and LPS (10μg/kg), respectively. At different times (6-12h) afteradministering the GalN/LPS, the DNA of the collected liver specimens was extractedfor analysis of DNA fragmentation. DNA ladder detection was employed as a clearinducer of hepatocyte apoptosis and used to determine the optimized doses ofGalN/LPS and the optimized time after the GalN/LPS injection. The results showedthat the liver apoptosis can be detected at6-8h after administering the GalN (400-700mg/kg) and LPS (10μg/kg), also indicated that the intraperitoneal injection of GalN (600-700mg/kg) and LPS (10μg/kg) for8hours is the optimized conditions ofhepatocyte apoptosis in mice.Then the hepatocyte apoptosis at8h after administering the GalN (700mg/kg)and LPS (10μg/kg) was systematically evaluated by histopathological analysis,TUNEL detection, flow cytometry and electron microscopy analysis. To clarify themechanisms involved in the process of the GalN/LPS-induced hepatocyte apoptosis,the expressions of tumor necrosis factor-α (TNF-α), transforming growth factor-β1(TGF-β1), caspase-3, Fas and Fas ligand (FasL) were determined by serumenzymeimmunoassay, immunohistochemical analysis and western blot analysis.The results indicated that strong apoptotic positive signals were observed byhistopathological analysis, DNA ladder detection, TUNEL detection, flow cytometryand electron microscopy analysis. And the expressions of tumor necrosis factor-α(TNF-α), transforming growth factor-β1(TGF-β1), caspase-3, Fas and Fas ligand(FasL) were significantly enhanced. In conclusion, Fas-mediated death receptorpathway could be the main mechanisms of the hepatocyte apoptosis induced byGalN/LPS. Furthermore, it is also probably associated with TGF-β-activated themitochondrial pathway.Part2The establishment of HL-7702hepatocyte apoptosis model and itsmechanisms involved in this processTo induce hepatocyte apoptosis by D-GalN in HL-7702hepatocytes, DNA ladderdetection was employed as the evaluation standard of hepatocyte apoptosis. Atdifferent times (4-12h) after HL-7702hepatocytes were incubated with the differentconcentrations of D-GalN (10-60mM), hepatocyte apoptosis was evaluated byanalysis of DNA fragmentation and caspase-3expression. In addition, hepatocyteinjury was also assessed by measuring the amount of AST and ALT leakage as wellas the MDA content and the SOD activity.The results indicated that typical apoptosis ladder was observed at6h after theHL-7702hepatocytes were incubated with60mM D-GalN. Also the caspase-3, AST,ALT and MDA levels of the D-GalN-sensitized hepatocytes were significantlyelevated. In conclusion, the incubation with60mM D-GalN for8hours may be theoptimized conditions of cell apoptosis induced by D-GalN in HL-7702hepatocytes. HL-7702hepatocyte apoptosis induced by D-GalN is probably associated with themitochondrial pathway.Part3Anti-apoptotic activity of natural compounds in HL-7702hepatocytesThe anti-apoptotic activities of three natural dicaffeoylquinic acids(3,4-O-dicaffeoylquinic acid,3,5-O-dicaffeoylquinic acid and4,5-O-dicaffeoylquinicacid) were evaluated using the D-GalN-induced apoptosis/injury model in HL-7702cells. Hepatocyte apoptotic changes were demonstrated by DNA ladder analysis,caspase-3activation and TGFβ1level. Oxymatrine at a concentration of200μg/mlwas used as a reference drug. The results showed that3,4-O-dicaffeoylquinic acid and4,5-O-dicaffeoylquinic acid significantly reduced the caspase-3levels atconcentrations of50μg/ml and100μg/ml, respectively.3,4-O-dicaffeoylquinic acidmarkedly inhibited the TGFβ1expression at concentrations of50μg/ml and100μg/ml. Also,4,5-O-dicaffeoylquinic acid at a concentration of50μg/ml significantlydeclined the TGFβ1level of the D-GalN-challenged hepatocytes. Oxymatrine, as thereference drug, showed the similar effect. In conclusion, the dicaffeoylquinic acidspossess the anti-apoptotic activity.In summary, this study established the D-GalN/LPS-induced hepatocyteapoptosis model in mice and the GalN-induced HL-7702hepatocytes apoptosis model,also investigated the mechanisms involved in the process of hepatocyte apoptosis.The mechanisms of the hepatocyte apoptosis induced by GalN/LPS is probablyassociated with Fas-mediated death receptor pathway and TGF-β-activated themitochondrial pathway. Furthermore, the present investigation also verifies thatnatural dicaffeoylquinic acids possess the anti-apoptotic effects. The establishment ofhepatocyte apoptosis model provides a powerful platform to clarify the pathogenicmechanism of hepatitis and screen anti-hepatitis drug.